The specific aim of this application is to develop a reproducible system in vitro aimed at examining the interaction between gingival fibroblasts and epithelial cells on the tooth surface. The long-term objectives are to use this system for elucidating basic biological processes that govern the formation of a new dentogingival junction. The soft tissue of premolar roots obtained from dogs will be removed and the roots will be planed and cut into 400 Mum thick root slices (RS) whose periphereal surface is cementum. The root slices will be sterilized in antibiotics and then conditioned in AlphaMEM + 15 FCS 24 h prior to their use. To obtain epithelial cells (EC), explants of rat attached gingiva will be placed on the cut surface of the root slices with the epithelial layer facing the tooth and will be maintained in culture. Epithelial cells migrate from the explant before gingival fibroblasts. The explants will be removed from the culture by trypsinisation and mechanical separation before the initiation of the fibroblastic migration. The appropriate time for doing this will be determined by SEM and TEM examination of cultures that will be harvested at 2,4,6,8 and 10 d after explanation. The rate of migration of epithelial cell on the cut surface and onto the peripheral surface of the RS's will be determined by SEM examination of specimens 1,2,4 and 6 d after the removal of the explants from the culture. Gingival fibroblasts (GF) have been shown to migrate from the floor of the culture dish onto the peripheral surface of RS's when these are placed on a confluent layer of GF's. The rate of migration of GF's on the cementum will be assessed by SEM examination of cultures harvested 2,4,6,8 and 10 h following the placement of RS's on confluent layers of gingival fibroblasts. Based upon the epithelial and fibroblastic rates of migration on the cementum, RS's bearing epithelial cells on their cut and peripheral surfaces will be placed on confluent layers of gingival fibroblasts and maintained in culture until the epithelial cells and the gingival fibroblasts will eventually meet on the cementum of the RS's. Using SEM and TEM techniques the following will be assessed: a) the time required for the epithelial cells to meet the gingival fibroblasts; b) whether or not one of the cell types will migrate over the other and if so which of them will maintain contact with the cementum; c) and the ultrastructure of the junction between the two cell types and between them and the cementum.